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Human Molecular Genetics, 2004, Vol. 13, No. 15 1551-1561
DOI: 10.1093/hmg/ddh178
Human Molecular Genetics, Vol. 13, No. 15 © Oxford University Press 2004; all rights reserved

Transgenic mouse models support HCR as an effector gene in the PSORS1 locus

Outi Elomaa1, Inkeri Majuri1, Sari Suomela2, Kati Asumalahti1,2, Hong Jiao4, Zahra Mirzaei4, Bjorn Rozell5, Karin Dahlman-Wright4, Johanna Pispa3, Juha Kere1,4,5,* and Ulpu Saarialho-Kere2,6

1Department of Medical Genetics, 2Department of Dermatology and 3Institute of Biotechnology, University of Helsinki, Helsinki University Central Hospital, Helsinki, Finland, 4Department of Biosciences at Novum, Karolinska Institutet, Huddinge, Sweden, 5Division of Clinical Research Center and Pathology, Department of Laboratory Medicine, Huddinge University Hospital and 6Karolinska Institutet at Stockholm Soder Hospital, Stockholm, Sweden

Received March 14, 2004; Revised May 1, 2004; Accepted May 25, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Genetic susceptibility for psoriasis is regulated to the greatest extent by the PSORS1 locus. Three psoriasis-associated susceptibility alleles have been identified within it, namely, HLACw6, HCR*WWCC and CDSN*5, but strong linkage disequilibrium between them has made it difficult to distinguish their individual genetic effects, and animal models to study their effects are not known. To study the function of HCR, we engineered transgenic mice with either a non-risk allele of HCR or the HCR*WWCC risk allele under the control of the cytokeratin-14 promoter. These choices were motivated by the apparently dominant effect of PSORS1 on psoriasis susceptibility and the physiological expression of HCR in basal keratinocytes. Transgenic mice appeared phenotypically normal and histologically their skin was indistinguishable from wild-type mice. Expression studies using Affymetrix arrays suggested that the HCR risk allele has specific functional consequences relevant to the pathogenesis of psoriasis. Comparison of gene expression changes between non-risk and risk allele mice revealed similarities to previous observations in human psoriatic skin, including upregulation of cytokeratins 6, 16 and 17 in risk allele mice. We also observed changes in the expression of genes associated with terminal differentiation and formation of the cornified cell envelope. Our results support the concept that HCR may constitute an essential gene in the PSORS1 locus. These observations are also compatible with a model that a susceptibility gene for psoriasis induces changes that are contributory but not sufficient by itself to produce the clinical phenotype.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Psoriasis is a common chronic skin disorder characterized by keratinocyte hyperproliferation, inflammatory infiltrates and neoangiogenesis, but its fundamental pathophysiology is still unknown. In several genetic association and linkage studies, the most important genomic region in psoriasis predisposition is PSORS1 near HLA-C in chromosome 6p.21 (1–5). Three strongly psoriasis-associated susceptibility alleles have been identified within it, namely, HLACw6, HCR*WWCC (alpha-helix coiled coil rod homolog) and CDSN*5 (corneodesmosin), but strong linkage disequilibrium between them has made it difficult to distinguish their individual genetic effects (1,6).

We have previously shown that the HCR gene is highly polymorphic and has a psoriasis-associated allele (HCR*WWCC) in different populations (1,7). The predicted structure of the risk allele of HCR protein differs from the wild-type allele by a shorter first alpha-helical domain, affecting possibly also the antigenicity or biochemical properties of the protein. HCR protein is differently expressed in lesional psoriatic skin compared with normal skin (1,8). In normal and non-lesional psoriatic skin, the protein appears in basal keratinocytes. In lesional psoriatic skin, HCR expression is enhanced within nuclei and cytoplasm not only basally but also suprabasally above dermal papillae, showing attenuated expression in other parts of the epidermis. Finally, staining for the cell proliferation marker Ki67 shows inverse pattern compared with HCR staining, suggesting that HCR may have a role in keratinocyte proliferation. All these findings make HCR a plausible effector gene in PSORS1 (1). With little homology to other known proteins, the physiological function of HCR in skin remains poorly understood.

The purpose of this study was to investigate the functional role of HCR using two transgenic mouse models, namely, mice expressing either a human non-risk allele of HCR or the HCR*WWCC risk allele under the control of the cytokeratin-14 (K14) promoter. These choices were motivated by the apparently dominant effect of PSORS1 on psoriasis susceptibility and the physiological expression of HCR in basal keratinocytes. The transgenic mice are phenotypically normal, but our gene array results from the skin of these mice suggest that the HCR risk allele has specific functional consequences relevant to the pathogenesis of psoriasis and support the concept that HCR may constitute an essential gene in the PSORS1 locus.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Generation of transgenic mice
As HCR protein is detected in keratinocytes in normal as well as in psoriatic skin, we generated transgenic mice that overexpress human HCR under the control of K14 promoter. The K14 promoter predominantly targets transgene expression to basal epidermal keratinocytes and to outer root sheath keratinocytes of hair follicles. To study whether there is a difference in function caused by the psoriasis-associated HCR*WWCC risk allele when compared with the common low-risk allele, we made two constructs where either the wild-type or the psoriasis-associated form of human HCR cDNA was placed under the K14 promoter (Fig. 1A). Multiple lines of transgenic mice were generated. Southern blot and PCR analyses of genomic DNA revealed transgene incorporation in 14 normal allele and in 17 risk allele mice out of 81 and 75 born mice, respectively. All transgenic mice were viable and fertile and displayed no obvious phenotypic abnormalities (Fig. 1B). Two normal allele and three risk allele founder mice were further analyzed and bred to the FVB/N strain to generate positive lines. Expression of transgenes was confirmed by RT–PCR of total RNA isolated from skin lysates of mice (data not shown). Expression was also verified by SDS–PAGE and western blotting of protein extracts from the skin of transgene positive animals (Fig. 1C). All transgenic lines overexpressed human HCR both on mRNA and on protein levels. The amount of endogenous murine Hcr was too low to be detected from skin lysate by western blotting (data not shown). To measure the expression level of the transgene in different lines, we performed quantitative RT–PCR (TaqMan analysis) from RNA extracted from mouse skin. For comparison, expression values of transgenic lines were normalized with the lowest expression value (expression of line 132 was set for 1). Normalized expression values for the non-risk lines 12 and 34 and for the risk allele lines 106, 128 and 132 were 3±1, 8±6, 1.2±1.7, 13±2.8 and 1±0.6, respectively (mean±SD between animals). Using t-test, the expression levels of transgenic HCR in groups of mice used for subsequent Affymetrix analyses were found to be not significantly different between the risk and non-risk allele transgenic mice by t-test. We also estimated copy doses of transgenes in different mouse lines from band intensities on Southern blots (data not shown). These results agreed well with those obtained from TaqMan analysis of transgene expression with one exception. Line 128 seemed to have fewer copies of transgene than lines 12 and 34, even though it showed the highest expression on mRNA level.



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  		Figure 1. Transgenic mice expressing non-risk or psoriasis-associated risk allele of HCR under the K14 promoter. (A) Schematic representation of K14–HCR transgene construct. The non-risk or the psoriasis-associated HCR*WWCC risk cDNA of human HCR was cloned into PG3Z–K14 vector using BamHI restriction site. Before cloning, additional SmaI and SacI sites were introduced to allow the recovery of linear K14–HCR fragment. The linearized transgenic fragments containing K14 promoter (K14), ß-globin (ßG) intron, either of the human HCR cDNAs and the polyA were released from the vector with SacI. Transgenic mice appear phenotypically normal (B). A mouse with HCR*WWCC expressed in the skin is shown. Expression of transgenes was confirmed by SDS–PAGE and western blotting of extracts from the skin of transgene positive animals (C). Western blots with the antibody against human HCR demonstrated that mice from non-risk lines 12 and 34 and from risk lines 106, 128 and 132 displayed a band of around 100 kDa corresponding to human HCR polypeptide, whereas a sample from the wild-type mouse was negative.

 
Histological analysis of the skin
Without environmental challenge, skin morphology appeared grossly normal and was indistinguishable between all transgenic and control mice by hematoxylin and eosin staining (Fig. 2A–C). When skin samples of transgenic mice were stained with antibodies against human HCR, expression of protein was detected in basal keratinocytes (Fig. 2D and E) as observed with the endogenous human HCR protein (1). We also carried out immunostainings with frozen sections of skin, but no additional staining was observed when compared with paraffin sections. Skin of wild-type mice was negative with the antibody against human HCR as expected (Fig. 2F). The intensity of immunostaining differed between various transgenic lines, agreeing with the results of RT–PCR. Variation in expression levels was also observed between individual mice belonging to the same transgenic. Routine health surveillance showed the mice to be free from infection by ectoparasites. Special stains (PAS) did not reveal fungal colonization. We also bred mice that were bilineally transgenic, but these mice did not show any abnormalities either. Immunohistological analyses with antibodies against markers commonly used for psoriatic skin did not reveal any differences between transgenic and wild-type mice. These markers included the proliferation marker Ki67, the differentation markers cytokeratin-5 and filaggrin and the T-lymphocyte marker CD3 (data not shown).



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  		Figure 2. Skin morphology and immunostaining for HCR in 5-week-old male K14–HCR transgenic mice. The skin appeared normal for both the non-risk (A) and risk transgene models (B) and was undistinguishable from the wild-type (C). Paraffin sections from the skin of transgenic mice with a non-risk (D) or risk (E) allele of HCR were stained with antibodies against human HCR. Expression of protein was detected in basal keratinocytes. Arrows point at cells positive for HCR. Wild-type mice show no staining for HCR (F). Scale bars: 36 µm, A–C; 12 µm, D–F.

 
Allele-specific gene expression changes in transgenic mice
To understand possible biochemical consequences of HCR expression in the skin, we prepared RNA samples from risk, non-risk and wild-type mice (four, four and three animals, respectively). To avoid lineage-specific artifacts, the transgenic mice were from four different lines (non-risk lines 12 and 34 and risk allele lines 106 and 132). The individual RNA samples were then analyzed by Affymetrix gene arrays to detect variations in gene expression levels. We used two methods to consider transcripts that were differentially expressed among the wild-type, non-risk and risk allele mice. We identified differentially expressed genes based on a combination of >2-fold changes and t-tests (P<0.001) and assayed whether the means of gene expression in any two groups were significantly different. A complete list of genes showing significant changes in expression in Affymetrix assays with transgenic mice can be found elsewhere (Supplementary Material, Tables S1–S5).

Altered gene expression between transgenic mice and wild-type mice
A comparison between transgenic mice and wild-type FVB mice revealed 43 upregulated genes in transgenic mice with ≥4-fold average change (Supplementary Material, Table S1). Selected skin-related genes are shown in Table 1. Among the most interesting are Mmp3 (matrix metalloproteinase 3), Mmp13 (matrix metalloproteinase 13), Btc (betacellulin, epidermal growth factor family member), Tnfrsf6 (tumor necrosis factor receptor superfamily, member 6), Tnfaip2 (Tnf alpha-induced protein 2), Col3a1 (procollagen, type III, alpha 1), Ltbp1 (latent transforming growth factor beta-binding protein 1) and Calm4 (calmodulin 4). Nine genes showed a consistent increase in expression levels in all eight transgenic mice (24 comparisons) (Supplementary Material, Table S2). Among them Krtdap (keratinocyte differentiation-associated factor) and Plxnb2 (plexin B2) showed >3-fold increase and Igfbp5 (insulin-like growth factor binding protein 5) a 2-fold increase (Table 1).


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  		Table 1. Selected skin-related genes showing changes in expression when comparing transgenic with wild-type mice (average values from 24 comparisons are shown)
 
Sixty-three downregulated genes were found with at least 4-fold change (Supplementary Material, Table S1). Highly significant downregulation of the following genes was detected in transgenic mice: several keratin-associated proteins and hair keratins, S100a3 (S100 calcium binding protein A3) and Fgf5 (fibroblast growth factor 5) (Table 1). Less significantly downregulated genes were Wnt11 (wingless-related MMTV integration site 11), Gjb2 and 6 (gap junction membrane channel proteins beta 2 and 6), Ctse (cathepsin E), Sprr2H (small proline-rich protein 2H), Tgm3 [transglutaminase 3, E polypeptide, cross-linking protein in the cornified envelope (CE)] and Dsc2 (desmocollin 2) (Table 1).

Altered expression between risk and non-risk allele lines
Different expression profiles between transgenic mice with risk alleles and with non-risk alleles were illustrated in two ways. One is a direct comparison between two transgenic groups and differential expression was indicated by >2-fold change. The other is based on t-test of expression levels adjusted by basal lines (expression of wild-type mice) and the threshold P-value 0.001.

When the array results of the risk mice were compared with the non-risk mice, 56 genes were 2-fold upregulated and 14 genes 3-fold (Supplementary Material, Table S3). Largest changes for upregulated genes in the risk allele mice were observed for several small proline-rich proteins, Sprr1B, 2A and 2D, Krt2-6a (keratin 6a) and its partner Krt1-16 (keratin 16) and Jak2 (Janus kinase 2) (Table 2). Less upregulated were serum serpine1 (Pai1, serine or cysteine proteinase inhibitor), amyloid A3, tenascin C, IL-6, Caspase 6, Sprr2H and 2F, Tnsfs11, MMP13, 9 and repetin (Table 2). When t-test was performed, the following genes showed statistically significant upregulation (P<0.001): Sp4 (trans-acting transcription factor 4), Sprr2F, Sprr1B, Cul3 (cullin 3), Zac1 (zinc finger protein regulator of apoptosis and cell cycle arrest), Clk4 (CDC-like kinase 4), Defb1 (defensin beta 1), Gpc1 (glypican 1), Peg3 (paternally expressed 3), Tank (TRAF family member-associated NF-{kappa}B activator), Krt2-6a (keratin 6a) and destrin (Supplementary Material, Table S4).


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  		Table 2. Selected skin-related genes showing significant change in expression when comparing mice with the risk allele of HCR with mice with non-risk allele
 
Eighty-three genes were 2-fold downregulated and 16 genes 3-fold downregulated when the risk mice were compared with the non-risk group. Most strongly downregulated genes in the risk allele mice compared with the non-risk mice were several keratin-associated proteins and acidic hair keratin 5 (Krt1-24) (Table 2). Less significantly downregulated genes were Krt1-1 (type I hair keratin), Krt2-18 (basic hair keratin 5), Krt1-19 (cytokeratin 19), Msx2 (homeo box, msh-like 2), Gal (galanin), S100a3 (hair cuticle-associated protein) and Mmp12 (matrix metalloproteinase 12) (Table 2). When t-test was performed, the following genes showed significant downregulation (P<0.001): serpin6 (serine or cysteine proteinase inhibitor, clade A, member 6), Cdh3 (cadherin 3), Ap1b1 (AP-1 beta subunit), Prkcz (protein kinase C zeta), Rxra (retinoid X receptor alpha), S100a13 (S100 calcium binding protein A13), Klf9 (Kruppel-like factor 9), Cdh1 (cadherin 1), Il6st (IL-6 signal transducer), Ubc (ubiquitin C) and S100a10 (S100 calcium binding protein A10) (calpactin) (Supplementary Material, Table S5). A comparison of these findings to genes related to psoriasis in previous studies is shown in Table 3.


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  		Table 3. Genes with altered expression in transgenic mice and human orthologs implicated in psoriasis
 
Pathway analysis of gene expression differences
Using the Biocarta net tools, changes of genes in different signal pathways (NF-{kappa}B signaling, keratinocyte differentiation, EGF, TGF-beta and IFN-gamma signaling pathways, cytokines and inflammatory response, cell-to-cell adhesion and cytoskeleton organization and biogenesis) relevant to psoriasis were examined. When P<0.01 (t-test) was taken as the criterion of significant change, the changes were detected in the cytoskeleton organization and biogenesis cluster in which keratin-6a (Krt2-6a) and 16 (Krt1-16) and alpha 2 actin were upregulated in the risk mice compared with non-risk mice.

Cluster analysis
Similarity of gene expression patterns was studied by cluster analyses. In addition to using the entire expression data set, we split it into several subgroups by key words such as integrin, interleukin, transcription factor, proteinase, binding protein, protease or keratin for tree construction. We also limited analyses to males only to reduce possible hormonal effects.

Out of seven two-way cluster trees, only the keratin group, containing 57 transcripts representing 37 genes, showed clearly distinct gene clusters, and the separation among risk, non-risk and wild-type mice was pronounced (Fig. 3). Six male mice classified keratin-related genes into two main groups. Twenty genes showed higher expression levels in risk mice when compared with the other two groups, and 14 genes showed lower expression levels in risk mice. Among several clusters, two were most obvious (Fig. 3, clusters 1 and 2). One interesting gene in cluster 2 was Krtdap (keratinocyte differentiation-associated protein), which was clearly differentially expressed according to the mouse group, namely, upregulated in risk mice compared with non-risk mice and downregulated in wild-type mice. In cluster 1, skin-related genes Krt2-5 (keratin 5), Krt2-6a (keratin 6a) and Krt1-17 (keratin 17) showed higher expression levels in risk mice compared with those in either non-risk or wild-type mice. On the other hand, most genes showing lower expression level in risk mice belong to hair keratins or keratin-associated proteins, known to be expressed mainly in hair. The unique clustering profile of keratin-related genes indicated the importance of these genes in classifying the different mouse groups and supported a role of HCR in cytoskeleton organization as suggested by pathway analysis.



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  		Figure 3. Cluster analysis of Affymetrix expression results. Two-way cluster trees were constructed on the expression results of six male mice. Wild-type animals (W1 and W2), transgenic mice with non-risk allele (N1 and N2) and transgenic mice with risk allele (R1 and R2) were studied. (A) Signal intensity values of 57 genes selected by the keyword ‘keratin’ show genotype-dependent clustering. Genes are annotated by their titles from Affymetrix annotation. For comparison, genes selected by the keyword ‘interleukin’ (B) or ‘integrin’ (C) do not show similar genotype-dependent clustering (gene names omitted). The normalized expression levels are indicated by color code. Red indicates upregulated genes, yellow is average and downregulated genes are in blue (color index bar on the left). Affymetrix data were verified by using quantitative RT–PCR (TaqMan) (D). Expression levels of Mmp9, S100a10 and keratin-6a was measured from the skin RNA isolated from wild-type (white columns), non-risk (gray) and risk (black) mice. Animals (three mice from each group) were from transgenic lines 12 (non-risk) and 128 (risk). Expression levels of Mmp9 and cytokeratin-6a was upregulated and the expression of S100a10 was downregulated in risk when compared with non-risk mice. Error bars: mean±SD.

 
Quantitative real-time PCR
Quantitative RT–PCR analysis is commonly used to verify expression data obtained from Affymetrix. We performed TaqMan analysis using RNA isolated from the skin of transgenic mice (the non-risk line 12 and the risk line 128) and wild-type mice to measure expression levels of Mmp9, S100a10 and cytokeratin-6a (Fig. 3D). According to our Affymetrix data, expression of Mmp9 and cytokeratin 6a were 2- and 4-fold upregulated, respectively. When the expression of these genes was compared using TaqMan analysis, similar changes of expression were observed as with Affymetrix assays (Fig. 3D). Using TaqMan, the decrease in expression of S100a10 in risk compared with non-risk mice was measured to be 2-fold. The amount of transgene mRNA in mice used for TaqMan did not seem to correlate with the expression of measured genes. This supports that the observed changes of expression are caused by allele-specific differences of transgenes and not merely by different transgene doses.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
This is the first study in which specific risk and non-risk alleles of a candidate gene for psoriasis susceptibility have been functionally examined in transgenic mouse lines. Overall, the HCR transgenic mice were phenotypically normal, but detailed analysis of gene expression in skin revealed genotype dependent allele-specific differences between the transgenic mice. We argue that this finding is strongly supportive of a functionally distinct role for the human psoriasis-associated HCR*WWCC allele. When assessed for similarities with previous observations in psoriatic skin (9), the results suggest many similarities, but no complete agreement between the HCR mouse models and human psoriasis (Table 3). These observations are compatible with a model that a susceptibility gene for psoriasis induces changes that are contributory but not sufficient by itself to produce the clinical phenotype. Additional genes and/or environmental triggers are known to be necessary to launch human psoriasis, because only 10–20% of those who carry the susceptibility allele for PSORS1 develop the clinical phenotype. There are no really good animal models for psoriasis (24), although several transgenic mouse lines recapitulating some features characteristic for psoriasis have been established, such as mice overexpressing amphiregulin under the K14 promoter (25), mice expressing beta 1 integrin alone or in combination with alpha 2 and 5 integrins under the involucrin promoter (26), mice with bone morphogenetic protein 6 under K10 promoter (27), mice overexpressing IL-1a (28), TGF-alpha (29) or IL-20 (30). However, many mouse strains overexpressing important cytokines implicated in the pathogenesis of psoriasis, such as IL-6, VEGF and IFN-gamma, have not demonstrated a psoriatic phenotype (31–33).

To study the function of HCR, we made transgenic mice expressing the non-risk allele or the psoriasis risk allele under the K14 promoter. As expected, HCR expression was seen in the basal layer of mouse skin where the K14 promoter normally targets it. There was no remarkable difference in the expression pattern of HCR protein between non-risk and risk allele mice. In both mouse groups, immunostaining for HCR protein was rather granular and perinuclear. Similar localization has also been observed for endogenous HCR in human epidermis (1,8). We also cultured keratinocytes isolated from skin of transgenic mice. Using immunofluorescence staining, granular HCR protein was detected not only perinuclearly but also throughout the cytoplasm and in pseudopodia (data not shown), agreeing with the recent results of Sugawara et al. (34), who localized HCR to endosomes and mitochondria. Sugawara et al. further suggested that one of the roles for HCR that they called (SBP) is to function in steroidogenesis interacting with steroidogenic acute regulatory (StAR) protein. Without any environmental challenge, the skin of transgenic HCR mice looked normal and was indistinguishable from the wild-type mice.

A number of changes in gene expression was observed when the array results of the risk mice were compared with the non-risk mice. Among the most interesting genes were keratins 6 and 16, previously found altered in psoriasis. In normal skin, they are associated with the outer root sheath of hair follicles and are not seen in interfollicular epidermis. In psoriatic skin, their expression is induced and they are used as markers for hyperproliferation of keratinocytes (27,14). When the array results of the risk mice were compared with the non-risk mice, cytokeratins 6 and 16 were upregulated. This agrees well with the gene array results of Bowcock et al. (9) in human psoriatic skin. Significant changes were also observed in genes of keratin-associated proteins, but they are mainly expressed in the hair fiber cortex and to lesser extent in the cuticle (35).

We performed cluster analysis for selected protein groups using data obtained with male mice. Interestingly, only keratin-related genes (genes selected with the keyword ‘keratin’) organized the mice in clusters according to their genotype. Expression of integrin-, interleukin-, transcription factor-, proteinase- or protease-related genes did not show any genotype-specific changes among the risk, non-risk mice and wild-type mice. The most interesting gene in keratin cluster was keratinocyte differentiation-associated protein (Krtdap), which was clearly upregulated in risk mice compared with non-risk mice and downregulated in wild-type mice. Krtdap is a novel protein associated with the stratification of the epithelium. It is upregulated in the suprabasal cell layers of embryonic and adult epidermis and its expression is strictly regulated during the process leading to differentiation of basal cells in the rat (36). The corresponding human gene KIPV467 resides in chromosome 19q13 (GenBank accession no. XM_371161.1). Interestingly, one of the PSORS susceptibility loci in the human also locates near this region on chromosome 19 (37). Other upregulated genes in risk mice in keratin cluster were keratins 5, 6a and 17. Keratin 17 is not present in healthy skin but its expression is induced in psoriasis (9,38). Keratin 5 is expressed in the mitotically active keratinocytes and is upregulated in psoriatic skin (39).

Largest changes for upregulated genes in risk allele mice were observed for several small proline-rich proteins, Sprr1B, 2A and 2D. SPRRs are components of the innermost cytoplasmic layer of the CE and serve as cross-linkers between loricrin molecules in orthokeratinizing epithelia contributing to CE rigidity and resistance to mechanical stress (40). Interestingly, also Bowcock et al. (9) found SPRRs 2A and 1B to be 5–8 fold upregulated in psoriatic lesional compared with normal skin. There are three transgenic mouse models with epidermal barrier dysfunction that show dysregulation of SPRR genes. These models are the mice deficient for loricrin (40), the mice overexpressing a tight junction molecule claudin 6 (41) and the mice with a targeted ablation of the transcription factor Kruppel-like factor 4 (Klf4) (42). In human, SPRR genes cluster on chromosome 1q21, which interestingly coincides with the PSORS4 locus (43). Other genes of the same locus, such as filaggrin, involucrin and loricrin, were not significantly upregulated in our array. However, expression of repetin and some other genes that are known to participate in the formation of the CE and to influence barrier function were upregulated when comparing the risk and non-risk mice. Hyperproliferation of keratinocytes is a typical feature in psoriasis. Interestingly, it is also known to be a typical compensatory response of adult epidermis that is severely compromised in barrier function (29).

We observed changes in the expression of genes S100 a3, a10 and a13. These genes were downregulated when risk allele mice were compared with the non-risk mice. S100 proteins are calcium-activated signaling proteins that interact with target proteins to modulate biological processes such as membrane-associated events. The function of these proteins is not well understood, but they have been proposed to play an important role in keratinocyte differentiation as well as in the pathogenesis of psoriasis (9,44).

Several members of the matrix metalloproteinase family were upregulated in the risk mice. Mmp3 was upregulated in both transgenic lines compared with the wild-type. Mmp13 and Mmp9 were induced in the risk allele mouse compared with the non-risk, whereas Mmp12 was downregulated. We have previously shown in human psoriasis that MMP1, 3 and 9 are upregulated in psoriatic lesions (15). When MMP1 is overexpressed in transgenic mouse skin, similar morphological hyperproliferative changes, for example psoriasis (acanthosis, hyperkeratosis and basal cell proliferation), are observed (45). In fact, MMPs represent novel targets of anti-psoriatic therapy because of their anti-angiogenic effects.

A number of changes in gene expression have been described previously in psoriasis, including upregulation of tenascin C (9,19), PAI-1 (46), and IL-6 (11,47). We also found increase in expression of these genes when the risk and non-risk mice were compared (Table 3). All these genes may have relevance for the pathogenesis of psoriasis.

The function of HCR in the epidermis still remains largely unknown and has only been highlighted based on its localization within the strongly psoriasis-associated locus PSORS1. It is expressed by keratinocytes in psoriasis in a specific manner compared with some other inflammatory skin diseases (1,8). Intriguingly, alleles of HCR that associate with human risk and non-risk haplotypes for psoriasis mediate allele-specific effects on the expression profiles of keratins and several genes associated with terminal differentiation and formation of the cornified cell envelope in mouse skin. These results may suggest a functional role for HCR in the PSORS1 locus and should motivate further studies to understand the detailed functions of the HCR protein.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENTARY MATERIAL
 REFERENCES
 
Transgenic constructs and production of transgenic mice
With the aim of understanding the functional role of HCR, we engineered two types of constructs to produce transgenic mice, using either the normal human allele of HCR or the psoriasis-associated HCR*WWCC risk allele under the control of the K14 promoter. Cloning of the human HCR corresponding to normal allele has been described elsewhere (1,7). Risk allele cDNA was obtained by reverse transcriptase PCR (RT–PCR) using RNA isolated from a skin biopsy of a psoriasis patient with the HCR*WWCC risk allele. The following primers were used for RT–PCR: forward primer containing a BamHI site (underlined) 5'-CGCGGATCCCTTTCAACTCTGCCAAGA-3' and reverse primer 5'-CCACCCACTTCTCCAGGAT-3'. Both cDNAs, normal and risk form of HCR were first cloned into the pCMV5 vector from which they were transferred to the K14 expression vector PG3Z–K14 cassette (48). Before transfer, the SphI and HindIII restriction sites at 3' side of the polyA in the pG3ZK14 (Fig. 1A) were used to introduce additional SmaI and SacI sites, to allow the recovery of linear K14–HCR fragment for pronuclear injection. For that purpose, a restriction cassette of 37 bp containing sites for SphI, SmaI, SacI and HindIII was synthesized using the oligonucleotides 5'-GCAGCATGCTGCCCGGGGAGAGCTCCTCAAGCTTCTT-3' and 3'-CGTCGTACGACGGGCCCCTCTCGAGGAGTTCGAAGAA-5' (restriction sites are underlined).

Using BamHI sites, HCR cDNAs, including the entire coding regions flanked by a 52 bp 5' and a 42 bp 3' untranslated sequences, were transferred from pCMV5 vector into pG3ZK14. The correct sequence and orientation of HCR inserts were verified by restriction mapping and direct sequencing. The linearized transgenic fragments containing K14 promoter, ß-globin intron, human HCR cDNAs and the polyA were released from the vector with SacI. Fragments were purified using agarose gel electrophoresis followed by gel extraction kit (Qiagen). The fragments were eluted using ultrapure buffer containing 10 mM Tris (pH 7.5) and 0.2 mM EDTA. The transgenes were microinjected into the pronuclei of fertilized oocytes of FVB/N mice (Jackson Laboratories, Bar Harbor, ME, USA). A total of 81 and 75 mice were produced for screening for the non-risk and risk allele constructs, respectively. K14–HCR founder mice were crossed to the FVB/N mice to establish a co-isogenic transgenic line. Tissue specimens for RNA, protein and histological analyses were collected at different ages (1, 2, 4, 6 and 12 months). All animal studies were approved by the regional committee for animal experiments at the University of Helsinki (license number STU 27 A/2002).

Screening of transgenic mice
Potential founders and littermates were screened for integration by PCR. Genomic DNA for screening was isolated from tails, digested in 40 mM Tris–HCl, pH 8.0, 20 mM EDTA, 200 mM NaCl, 0.5% SDS, 0.5% ß-mercaptoethanol and 20 mg/ml proteinase K at 60°C overnight, precipitated with isopropanol, washed with ethanol and dissolved in water. Samples were analyzed by PCR using a forward primer annealing to K14 sequence 5'-ACATCCTGGTCATCATCCTGCC-3' and a reverse primer specific to HCR sequence 5'-CTAGCCGCCTCTCTGAGACATC-3'. The PCR result of founder animals was verified by Southern blotting using a K14 promoter specific fragment of 795 bp as a probe. The K14 fragment was generated by PCR using a forward primer 5'-AAGCCTGGGCAATAACAATG-3' and a reverse primer 5'-GAAAGCCCAAAACACTCCAA-3'. Southern blotting was performed using standard protocols. Blots were visualized by autoradiography.

Protein expression of transgenes was verified by western blotting. For western blot analyses, mouse skin was frozen in liquid nitrogen and subsequently stored at –70°C. Tissues were ground with a mortar and pestle in the presence of liquid nitrogen. Tissue powders were homogenized into Laemmli buffer using a syringe with needle and boiled for 3 min. Solutions were centrifuged for 10 min at 4°C. A total of 10–40 µg of protein per sample was analyzed by SDS–PAGE in the presence of ß-mercaptoethanol. Western blot analysis was carried out as described using 7.5% poly-acryamide gels and a rabbit anti-HCR serum for detection. Signals were detected with chemiluminescence (ECL, Amersham Pharmacia).

Immunohistochemistry
Samples of epidermis were taken from dorsal and ventral truncal skin, ear, tail and footpad. Tissue samples were fixed in 10% formalin solution, embedded in paraffin and sectioned. For histological analysis the sections were stained with hemotoxylin and eosin. For immunodetection of human HCR protein in transgenic mouse skin, tissue sections were treated as described previously for localization of HCR in human skin using affinity purified rabbit anti-HCR IgG (7 µg/ml), (1). The following panel of antibodies were used as markers in immunostaining: polyclonal antibodies to the proliferation marker Ki67 (Novocastra), mouse cytokeratin 5 (Berkeley Antibody Company), filaggrin (Berkeley Antibody Company) and T-lymphocyte marker CD3 (Novocastra, UK).

Immunostainings were performed using the avidin–biotin–peroxidase complex technique (Vectastain ABC Kit, Vector laboratories, Inc., Burlingame, CA, USA for HCR; StreptABComplex/HRP Duet (Mouse/Rabbit) Kit, DAKO, A/S Glostrup, Denmark, no. K0492 for Ki67). Aminoethylcarbazole (AEC) was used as a chromogenic substrate. Sections were pretreated with trypsin (10 mg/ml; HCR) or by microwaving in citrate buffer (Ki67). The tissues were counterstained with hemotoxylin. Controls for HCR were performed with preimmune sera (10 µg/ml) or normal rabbit immunoglobulin.

Preparation of cRNA for microarray analysis
Individual total RNA samples from dorsal skin of all three groups (three wild-type FVB animals, four non-risk and four risk allele animals) were used for microarray hybridization. Animals were from lines 12, 34, 106 and 132. Mouse skin was washed with 70% ethanol and a piece of ~2 cm2 from the back skin was cut for RNA isolation. The samples were rinsed with 70% ethanol and phosphate-buffered saline and frozen in liquid nitrogen. RNA was isolated using TRIzol (Invitrogen) according to the manufacturer's recommendations. Isolated RNA was resuspended in water and further purified using the RNAeasy total RNA isolation kit (Miniprep-Kit Qiagen, Chatsworth, CA, USA) according to the manufacturer's instructions. The RNA samples were treated with DNAse (Qiagen) to eliminate genomic DNA contamination. The quality of RNA was checked by spectrophotometry (A260/280 ratio), agarose gel eletrophoresis and by the Agilent Bio-analyzer (Agilent Technologies Inc.).

Double-stranded cDNA was synthesized from total RNA, and the cDNA was used as a template in in vitro transcription to generate biotinylated cRNA. Eight micrograms of labeled cRNAs were hybridized to each array, and scanning was performed after biotin–avidin–phycoerythrin amplification.

Affymetrix analysis
The microarray used in this study was U74Av2 murine genome Genechip probe array (Affymetrix Inc., Santa Clara, CA, USA) that contains probe sets representing ~12 000 genes (6000 well-characterized genes and 6000 expressed sequence tags, ESTs). The labeled target cRNA was fragmented, and hybridized to probe arrays according to Affymetrix expression analysis technical manual, P/N 700218 rev.2. The probe arrays were washed and stained. GeneChip 3.2 software (Affymetrix Inc.) was used to scan the images, to convert intensities to a numerical format and to obtain an average difference value for each probe in the array. Standard protocols provided by Affymetrix Inc. were employed for experimental procedure.

Data analyses
For construction of gene expression profiles, scanned output files were analyzed with the Affymetrix Microarray Suite 5.0 (Affymetrix Inc.). Single array analysis and comparison analysis were used to build databases of gene expression profiles (presented as absolute signal) and estimates of changes in gene expression (presented as signal log ratio).

Each chip was normalized for all probe sets to target intensity 100 to minimize discrepancies between an experiment and baseline array due to experimental variations to allow intermarry comparisons. Comparisons were performed between eight transgenic mice and three wild-type mice as well as between the two types of transgenic lines (risk allele versus non-risk allele). Twenty-four and 16 comparisons were formed from each of the above comparisons for further analysis.

Differentially expressed genes were defined based on t-test (P≤0.001) and fold changes were determined by using average values of signal log ratios (absolute value ≥1). We excluded genes if their expression, as defined by the Affymetrix software, was absent in all 11 arrays or if there was no change detected in all comparisons.

To classify genes in a similar manner and to see how well those genes can characterize different mouse lines, cluster analysis was performed using GeneSpring 5.1 software. (Silicon Genetics, Redwood City, CA, USA). Two-dimension hierarchical trees were built based on signal intensity values from single array analysis of Microarray Suite after normalization. Similarity of clusters is measured by standard correlation. Subgroups of genes were used for cluster analysis to focus on specific biopathways.

Related biological pathways were selected from BioCarta (http://www.biocarta.com/index/.asp) for survey, and altered biological processes were determined by changed gene expression profiles according to t-test (P≤0.001). Information on human orthologs was obtained through Affymetrix annotation tools (http://www.affymetrix.com/analysis/download_center.affx, March 2003).

Quantitative real-time PCR
Quantitative real-time PCR (TaqMan) was performed to confirm microarray results of the following murine genes: keratin-6a, Mmp9 and S100a10. Predesigned primer and probe sets for keratin-6a, Mmp9 and S100a10 (Assay-on-Demand gene expression product, Applied Biosystems) were used according to manufacturer's protocols. Rodent Gadph labeled with VIC reporter dye (Predeveloped TaqMan assay reagents, Applied Biosystems) was used as an endogenous control gene.

Total RNA was isolated from skin samples using the RNAeasy total RNA isolation kit (Miniprep-Kit Qiagen) according to manufacturer's instructions. RNA was reverse transcribed to cDNA (TaqMan Reverse Transcription Reagents, Applied Biosystems) and used as a template in a PCR. Real-time quantitative PCR assays were performed with the ABI PRISM 7700 sequence detector system (Applied Biosystems). Reaction conditions were programed on a power Macintosh 7200, linked directly to the sequence detector. PCR amplifications were performed according to manufacturer's recommendations.


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
REFERENCES
 
Supplementary Material is available at HMG Online.


   ACKNOWLEDGEMENTS
 
We wish to thank Irma Thesleff, Heikki Rauvala and Raija Ikonen from the Institute of Biotechnology, University of Helsinki, for advice and technical help in the production of the transgenic animals and Johanna Lahtinen for her excellent technical assistance. This study was supported by Academy of Finland, Sigrid Jusélius Foundation and the Helsinki University Hospital research funds.


   FOOTNOTES
 
* To whom correspondence should be addressed at: Department of Biosciences at Novum, Karolinska Institutet, 14157 Huddinge, Sweden. Tel: +46 734213550; Fax: +46 87745538; Email: juha.kere{at}biosci.ki.se


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 

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